microRNAs (miRs) naturally fine-tune the responsiveness of signaling pathways, and a number of miRs are known to play important roles in heart development and disease. To date, knowledge of miRs involved in any biological process comes primarily from expression profiling. Relatively few miRs have been tested for actual regulatory roles, therefore our current knowledge of both the prevalence, importance and actual function of miRs in biological control remains rudimentary. Our data show that it is feasible to apply high throughput technology to screen stem and progenitor cell-based assays against whole genome libraries of miR oligonucleotide mimics. Using this approach, we discovered miRs that play unanticipated roles in initiating embryonic heart formation and act by distinguishing cardiac mesoderm from foregut endoderm. Here we propose to apply this same approach to identify miRs that control the next step in cardiogenesis: the differentiation and diversification cardiomyocytes, smooth muscle, and vascular endothelial cells from a common progenitor. Although the proper diversification of cardiopoietic lineages is critical to heart formation, regeneration and disease, little is known about how the process is controlled. To address this question, we will apply whole genome miR library screening, followed by an iterative process of validation of individual miRs and targets and model building to yield a refined network of signaling proteins and miRs that control cardiopoiesis. The network of miRs and target proteins will be evaluated through targeted gain and loss of function studies in embryos to visualize how miRs regulate signaling proteins in order to guide cardiopoiesis. This research will yield two outcomes: First, the functional screening and subsequent in vivo testing will reveal miRs that control cardiopoiesis and hence be important for development, disease and regeneration. Second, we will construct and test signaling networks composed of the miR targets that will offer insight into the systems-level control of cardiopoiesis. The research is innovative in that it merges state of the art functional screening of miRs with miR target identification and systems analysis in order to expose the logic underying the emergence of distinct cell types in the heart from stem cells. PUBLIC HEALTH RELEVANCE: microRNAs have emerged as key regulators of many cellular processes, including heart development and disease. However, relatively few microRNAs have been functionally tested, so appreciation of the prevalence and importance of microRNA control of cardiac biology and physiology remains fragmentary. Here we will apply high throughput drug screening technology to discover microRNAs and target proteins that control heart development, revealing how diverse types of heart cells from stem and progenitor cells.